(1) Field of the Invention
The present invention relates to therapy of the immune system. In particular, the present invention relates to a primary cell derived biologic and methods of using the same to modify potentiation of the immune system.
(2) Description of Related Art
In a functioning and competent immune system, immature dendritic cells ingest antigens and migrate to the lymph nodes, where they mature. The resulting mature dendritic cells are then able to activate naïve T cells, creating antigen-specific cytotoxic T cells that then proliferate, enter the circulation, and search out and kill the antigenic target. This is generally a powerful, effective, and fast response. For example, the immune system is able to clear out an influenza infection between 7-12 days.
Antigenic targets can only be eliminated if the immune system is competent. Tumors and various other antigenic targets have effectively evolved strategies to successfully evade the host immune system, and various molecular and cellular mechanisms responsible for tumor evasion have been identified. Some of these mechanisms target immune antitumor effector cells. For example, dysfunction and apoptosis of these cells in the tumor-bearing host creates an immune imbalance that cannot be corrected by immunotherapies aimed only at activation of anti-tumor immune responses.
Apoptosis, or Type I cell death, is a type of programmed cell death and can be induced by stress, infection, or DNA damage. Apoptosis is an integral process during development; however, in certain instances it can actually do harm. For example, apoptosis of lymphocyte/hematopoietic populations, including T cells, can be a serious problem during cancer therapy-related chemotherapy and/or radiation therapy. These cells tend to be sensitive to chemotherapy and radiation therapy.
There are two major mechanisms controlling apoptosis in the cell, the p53 pathway (pro-apoptotic) and the nuclear factor kappa B (NF-κB) pathway (anti-apoptotic). Both pathways are frequently deregulated in tumors, as p53 is usually lost, while NF-κB becomes constitutively active.
Tumor-induced apoptosis of lymphocytes is thought to play a significant role in the immune suppression seen in cancer patients. Apoptosis of anti-tumor effector cells has been associated with expression of FasL on the surface of tumor cells. This is based on well-documented evidence that Fas/FasL interactions play an important role in the down-modulation of immune functions, including triggering of activation-induced cell death (AICD), to maintain central and peripheral tolerance. Many human tumors express FasL and can eliminate activated Fas+ effector lymphocytes via the Fas/FasL pathway. FasL expression on tumor cells has been shown to negatively correlate with patient prognosis. In addition, it has been shown that tumors can release membrane-associated FasL through secretion of membranous microvesicles (MVs), thereby providing an explanation for spontaneous apoptosis of T lymphocytes observed in the peripheral circulation of patients with cancer.
Applicants previously showed that MV detected in sera of patients with oral carcinoma induced caspase-3 cleavage, DNA-fragmentation, cytochrome c release, loss of mitochondrial membrane potential (MMP) and TCR ζ-chain down-regulation in activated T lymphocytes. Furthermore, Applicants demonstrated that these tumor-derived MV are distinguishable from immune cell-derived MV by their unique molecular profile and immune-suppressive properties. Recent data also indicate that MVs are present in sera of patients with squamous cell carcinoma of head and neck (H&NSCC) and that these MVs contain biologically active FasL which may be involved in mediating lysis of Fas positive T cells in the peripheral circulation. Thus, the activity of tumor-derived MV might significantly contribute to the dysfunction and death of effector T cells in cancer patients. The loss of these cells could be responsible for inadequate anti-tumor function and, by extension, inadequate immune responses to cancer vaccines.
It has been convincingly demonstrated that H&NSCC is able to induce functional defects and apoptosis in immune effector cells as well as antigen-presenting cells (APCs) by various mechanisms. In previous studies, Applicants have observed a high level of apoptosis of tumor-infiltrating lymphocytes (TIL) and T lymphocytes in the peripheral circulation of H&NSCC and melanoma patients. Applicants demonstrated that CD8+ T cells are more sensitive to apoptosis than CD4+ cells, and that the effector and tumor-specific subpopulations of CD8+ T cells are preferentially targeted for apoptosis. Also, individuals with HIV generally experience immune suppression caused by dramatic reductions in helper T cell populations. This reduction is caused by apoptosis of the HIV-infected helper T cells.
The mechanisms responsible for immune cell dysfunction in patients with cancer are numerous and varied. In addition to a wide variety of soluble immunosuppressive factors such as PGE2, TGF-β, IL-10, and VEGF, and pro-apoptic ligands such as FasL (described above) that are released by tumor cells or other cells in the tumor microenvironment, suppressor cell populations, i.e., regulatory T cells (T regs), have been shown to play a key role in down-regulation of anti-tumor host immunity.
Collectively, these mechanisms create a poisonous environment, which explains the failure of immunotherapy approaches in the past. In order to have an effective therapeutic outcome, these tumor-induced mechanisms of immune suppression must be directly addressed. With the newfound knowledge of the multiple causes of immune dysfunction seen in cancer patients, it is becoming more apparent that multiple active components are needed to create an effective cancer immunotherapy. However, there have been many difficulties in finding an effective immunotherapy and understanding its mechanism of action.
Since toxin-induced tumor regressions of human cancer achieved by William Coley early in the 20th century, cancer therapists have employed hundreds of different immune therapies with only relatively rare clinical responses. Because there was little or no insight into the cause of these failures, no consistent mechanism of action emerged. In order to establish a clear mechanism of action, a therapy needed to be devised which could consistently produce a response that could then be dissected.
Head and neck squamous cell cancer (H&NSCC) offers a good model since much is known about the immune defects seen in these patients. They include, to name a few, (Whiteside, 2001; Hadden, 1995): 1) T lymphocyte anergy and depletion induced by tumor and host-mediated mechanism including prostaglandins, T regs, myeloid suppressor cells, antigen-antibody complexes, and cytokines such as IL-10; 2) monocyte/macrophage functional defects with evidence of suppressor and inflammatory changes (Mantovani, 2002); and 3) dendritic cell (DC) defects characterized by sinus histiocytosis (SH) (Dunn, 2005).
Effective therapeutic efforts were needed to reverse these multiple defects. An extensive review of the literature (Hadden, 1995) and a series of pre-clinical experiments resulted in the primary cell-derived biologic (also known as IRX-2) protocol. The IRX-2 protocol, shown in FIG. 1, employs an initial dose of low dose cyclophosphamide (CY) (300 mg/m2) by intravenous infusion to reverse suppression by T regs and perhaps other forms of suppressors. The CY is followed by 10-20 daily injections of IRX-2 at the base of the skull to feed into the jugular chains of lymph nodes regional to the cancer.
IRX-2 was originally thought to act via increasing T lymphocyte number and function. Recent evidence indicates that reversal of tumor-induced apoptosis is also a major mechanism, as disclosed in U.S. Provisional Patent Application No. 60/990,759 to Signorelli, et al. Indomethacin (INDO) was administered daily for approximately 21 days to block prostaglandin production by tumor and monocyte/macrophages, a known cancer related suppression mechanism. Zinc was also administered as another aspect of the immunorestorative component of the strategy (Hadden, 1995).
Additionally, at the time the protocol was developed, the critical role played by dendritic cells as presenters of tumor antigen to T cells was unknown. It was also unknown that sinus histiocytosis (SH) reflected a DC defect, and specifically a tumor induced failure of maturation and antigen presentation. Mechanism of action studies disclosed in U.S. Pat. Nos. 6,977,072 and 7,153,499 to Applicants made it clear that the IRX-2 protocol reverses this DC defect and produces changes in regional lymph nodes which reflect a potent immunization (Meneses, 2003). More specifically, these patents disclose a method of inducing the production of naïve T cells and restoring T cell immunity by administration of IRX-2, which preferably includes the cytokines IL-1β, IL-2, IL-6, IL-8, INF-γ, and TNF-α. This was one of the first showings that adult humans can generate naïve T cells through molecular therapy. The presence of naïve T cells available for antigen presentation was important in the restoration of immunity.
The mechanistic hypothesis that underpins IRX-2 is similar to that of a therapeutic cancer vaccine, although no exogenous antigen is required. When administered into the neck, the agent is thought to act in the cervical lymph node chain directly on DCs to promote their maturation and subsequent ability to present endogenous tumor antigen to naïve T cells.
Non-clinical data regarding the mechanism of action of IRX-2 has shown that the agent effectively stimulates and activates human monocyte-derived DCs (Egan, 2007). IRX-2 treatment of immature DCs increased expression of CD83 and CCR7 (markers for maturation and lymph node migration, respectively), as well as differentiation molecules that are important for antigen presentation to naïve T cells. Additionally, IRX-2 induces CD40, CD54, and CD86, which are co-stimulatory receptors that are critical for activation of naïve T cells. Functional changes in IRX-2-treated DCs included an increase in antigen presentation and T cell activity. Taken collectively, IRX-2 treatment of immature DC drives morphologic, phenotypic, and functional changes that are consistent with the development of mature and activated DCs that are able to effectively stimulate naïve T cells.
In contrast to defined antigen-based therapeutic cancer vaccines where antigen-specific reactivity can be measured, rejection antigens have not been discovered in H&NSCC, thus limiting the ability to measure antigen-specific reactivity after IRX-2 therapy.
While IRX-2 was shown to increase T lymphocyte function and generate new immature T cells, there was no disclosure or suggestion and thus no conclusive demonstration that IRX-2 prevented apoptosis of those T cells once generated and it was not known what the function of the T cells were after presentation of antigen. There were no experimental results that showed that apoptosis of T cells was prevented or would even suggest the mechanism of action. Proliferation and apoptosis are separate cellular processes and it would be imprudent to assume that a factor that causes proliferation would necessarily protect from programmed cell death. The exact mechanism by which IRX-2 restores the antitumor response of T cells, and prevents their apoptosis, was neither expressly nor inherently disclosed in the prior art. Furthermore, while IRX-2 was shown to be effective in the mechanisms described above during cancer treatment, there has been no evidence that IRX-2 provides the same mechanism of action in other instances of immune suppression besides cancer.
Not only have individual cytokines not been able to completely restore each part of the immune system through the promotion of DC maturation, the generation of new T cells, and prevention of their apoptosis; but other therapeutics including multiple cytokines have not been able to do this as well. For example, MULTIKINE® (Cel-Sci) is effective only on the tumor itself, affecting the cell cycle of the tumor cells. PROVENGE® (sipuleucel-T, Dendreon), GVAX® (Cell Genesys), PROMUNE® (Coley Pharmaceutical Group), Dynavax TLR 9 ISS, ONCOPHAGE® (vitespen, Antigenics), CANVAXIN® (CancerVax), and TROVAX® (Oxford BioMedica) have been able to show antigen amplification, dendritic cell processing, and some cellular adjuvancy. TREMELIMUMAB® (Pfizer) and IPILIMUMAB® (Medarex and Bristol-Myers Squibb) only target the T regulatory cell population.
In addition, some therapeutic agents have addressed the issue of apoptosis of cells. There are several biological agents and small molecules that have been developed to prevent cellular and lymphocyte apoptosis. For example, International Patent Application Publication WO/2006/039545 to Maxim Pharmaceuticals, Inc. discloses the administration of a PARP-1 inhibitor and additionally an inhibitor of reactive oxygen metabolite (ROM) production or release to protect tumorcidal lymphocytes, including cytotoxic T lymphocytes and NK cells, from apoptosis. A cytotoxic lymphocyte stimulatory composition including various cytokines can be co-administered. This application reports that free radicals produced by tumor adjacent phagocytes cause dysfunction and apoptosis in tumorcidal or cytotoxic lymphocytes.
International Patent Application Publication WO/2005/056041 to Cleveland Clinic Foundation discloses latent TGF-β as a compound that can be used to protect a patient from treatments that induce apoptosis. The latent TGF-β induces NF-κB activity, thus preventing apoptosis.
International Patent Application Publication WO/2007/060524 to Fundacion de la Comunidad Valenciana discloses various ringed compounds that are inhibitors of Apaf-1 and therefore act as apoptosis inhibitors. Apaf-1 is an apoptotic protease-activating factor that makes up part of an apoptosome. Capsase-9 is activated within the apoptosome and initiates apoptotic signals.
Amifostine (ETHYOL, MedImmune) is another compound that is administered in order to reduce toxicities resulting from chemotherapy and radiotherapy. More specifically, it is an intravenous organic thiophosphate cytoprotective agent.
There are several disadvantages to these present treatments. For biological agents, there is the problem of difficulty in manufacturing and possible difficulty in specifically targeting a given cell population. For small molecules, there may be a problem of toxicity if used systemically. Further, agents with a single mechanism of action have shown a lack of efficacy because multiple activities are needed to promote anti-apoptotic effects in lymphocyte cell populations. Also, none of these treatments directly address the immunosuppressive environment created by the tumor. Thus, effective adjuvants and approaches to neutralize the tumor-induced suppression are lacking in the prior art.
In essence, the earlier work of Applicants described the mechanism of action of the primary cell derived biologic with respect to DC maturation and generation of naïve T cells, i.e. several specific levels of affecting the immune system. Presented herein is evidence of another level of effect, of the primary cell derived biologic, namely promotion of the survival of lymphocytes. The data herein, taken together with prior disclosures by the Applicants, show that the primary cell derived biologic has a corrective and positive effect on the generation and activation of specific effectors, and their subsequent survival—each level of the immune system, i.e. each arm of the immune system. Compositions of the prior art are directed to only one of these levels.
Therefore, there is a need for a composition that can effectively enhance both effector generation and effector survival and target each arm of the immune system to restore the immune system and provide a complete mechanism of action against immune suppression.